Vitamin D is initially hydroxylated at C - 25, i.e., 25-hydroxyvitamin D. However, its action to promote intestinal calcium absorption and bone mineral mobilization requires it's metabolic activiation via acquisition of a C-1 hydroxyl grouping, i.e., 1,25-dihydroxyvitamin D. This later process is catalyzed by a kidney mitochondrial enzyme system whose activity is regulated to meet the cellular needs for calcium and phosphorus. Our studies are directed toward delineating the molecular mechanism whereby metabolites of 25-hydroxyvitamin D act to feedback regulate the kidney activation process. A chick kidney tubule model will be used to determine the molecular requirements for C-1 and/or C-24 hydroxyl groupings in 25-hydroxyvitamin D metabolites which act on the kidney to suppress 1-hydroxylase and induce 24-hydroxylase enzyme activities. Metabolites who have their C-1 and/or C-24 functionalized with fluorine are incapable of being hydroxylated at C-1 or C-24. Therefore, these metabolites will be used as molecular probes to see if an hydroxyl group at C-1 and/or C-24 is required for the 25-hydroxy metabolites to regulate the 1- and 24-hydroxylase enzymes. Subsequent studies are directed toward: 1) isolating the flavoprotein reductase component of the hydroxylase enzymes and 2) delineating the mechanism whereby the rapid regulation, i.e., within 2 to 4 hr. of the 1- and 24-hydroxylase enzymes is accomplished. It is expected that results from our studies will be used to improve the preventive and therapeutic approaches to calcium and phosphorus metabolic diseases.